Identification of paediatric Hodgkin lymphoma biomarkers and novel therapeutic targets.

Although classical Hodgkin Lymphoma (CHL) in paediatric patients has a good
prognosis, the outcome is associated with a substantial proportion of
treatment-related toxicity and still about 10-20% of the patients progress
during or relapse after treatment. Strikingly, therapeutic regimens have not
changed much during the past decades. Current treatment protocols rely on
chemo- and radiotherapy, whereby patients are classified at diagnosis into
three different treatment groups based on a clinical staging system.
Radiotherapy can be omitted based on Fluoro-Deoxyglucose-Positron emission
tomography CT (PET-CT) treatment response.
HL is considered an immunological disease, where reactive cells in the tumor
microenvironment greatly outnumber malignant Hodgkin- and Reed-Sternberg (HRS)
cells. The microenvironment supports proliferation and survival of HRS cells.
Due to active crosstalk between HRS cells and their microenvironment, serum
biomarkers should be detectable and may be a surrogate for lymphoma viability.
HL biology impedes development of in vitro and in vivo assays for functional
studies to discover new therapeutics. Genetic analysis of malignant Hodgkin
cells has been hampered by their scarcity and has largely been done with
laser-dissected samples. In addition, apart from a clinical staging system at
diagnosis, there have been no prognostic molecular markers to stratify patients
into different therapy groups. Taken together this calls for efforts to
identify biomarkers and get an in-depth understanding of HL immunology and
biology to discover new therapeutic targets en less toxic therapies.

The aim of this project is to identify biomarkers and novel therapeutic targets
for pediatric Hodgkin lymphoma. To achieve this aim we defined three objectives:

1.Hodgkin Reed-Sternberg cells: To perform whole exome sequencing and gene
expression arrays of FACS-sorted malignant Hodgkin and Reed-Sternberg cells. To
get insights in the genetic profile of HRS cells and possibly translate this
into feature therapeutic targets.

2. Tissue microenvironment: To investigate the tumor microenvironment by
immunohistochemistry and gene expression profiling of microenvironment-derived
T-cells. Hereby identification and validation of new markers (TARC, PD-1). This
will possibly translate into novel therapeutic targets, for example PD-1-
blocking antibody.

3. Serum biomarkers: To investigate the value of serum TARC, other biomarkers
and ctDNA (see table 1 of the protocol) as disease response markers after each
cycle of chemotherapy and directly compare it to PET-scans, which is used for
response assessment in the current protocol. Analysing of serum samples after
treatment and during follow-up to investigate its value as markers for disease
recurrence.

In this multi-centre study, patients will be recruited from 5 paediatric
oncology centers in the Netherlands. Based on the incidence of HL, we expect to
include 30-40 patients with HL per year. More patients are expected to be
included due to an extensive collaboration with centres outside the Netherlands
within the European Network of Paediatric Hodgkin`s Lymphoma (EuroNet-PHL)
community; we expect to include ~60-80 patients per year.
Sampling will be performed by trained research nurses and doctors after
obtaining informed consent. All cases will be defined as cHL morphologically
and immunohistochemically and classified into histologic subtypes by an
hematopathologist. Whole exome sequencing of the malignant HRS cells will be
done in the laboratory of Prof. F. Holstege at the Princess Maxima Center,
Utrecht. In newly diagnosed patients or in patients at relapse or at
progression during treatment, immunohistochemistry for TARC will be performed
to confirm expression of TARC by Hodgkin tumour cells. Subsequently, T-cell
subpopulations, NK-cells, macrophages, PD-L1/ PD-1 expression and immune
inhibitory cytokines such as IL1-10 and TGF will be examined on tissue samples.
Serial serum samples will be measured at diagnosis (baseline) and at fixed time
points during treatment and follow-up (see table 2,3 and 4 in the protocol).
The biomarkers will be measured by Luminex® technology or by enzyme-linked
immunosorbent assay (ELISA) performed by the Laboratory of Translational
Immunology of the WKZ in Utrecht. Normal cytokine levels were defined on the
basis of the ELISA kit purchasers instructions. In addition, serum samples are
collected from age-matched healthy controls to define normal serum TARC levels
in paediatric patients. Biomarker levels will be directly compared to
PET-scans.cfDNA will be measured by different sequencing techniques and
compared with whole genome sequencing results of sorted Hodgkin cells. This
will be done at the research laboratory of Princess Máxima Center of Paediatric
Oncology, Utrecht

There will be no extra burden or risk for the patients.
We will take blood samples of on fixed time points before, during and after
treatment. In table 2,3 and 4 of the study protocol the exact time points are
mentioned, this depends on the treatment level conform the EuroNet-PHL
protocol. Blood samples will always be taken together with standard blood tests
according to the protocol. Two extra tubes of blood will be collected for this
research.
Moreover we will use some of the lymph node tissue that is taken out at
diagnosis with the biopsy to investigate expression of the biomarkers. This
changes nothing in the procedure of the biopsy.
For the control group, Two extra tubes of blood will be collected during the
already planned necessary blood tests during their outpatient clinical visit.